The objective of this proposal is to develop a biochemical-genetic approach to study the molecular mechanisms of the receptor-mediated endocytosis of transferrin by mammalian cells. Transferrin binds to receptors present on most cells, aggregates into clusters on the membrane exterior, and is internalized by endocytosis. We propose to isolated and study mutants deficient in the uptake process by exposing cells to a hybrid toxin whose cytotoxic activity depends on transferrin internalization. There are four specific aims in the project. First, the hybrid toxin will be made by coupling CRM 45, a structural variant of diphtheria toxin, to transferrin. CRM 45 is not ordinarily cytotoxic but should become so when carried into the vesicular compartment of the cell by transferrin. Second, we will characterize the mechanism by which the hybrid toxin acts. It should mimic the intoxication mechanism of diphtheria toxin, which is known to require endocytosis and acidification of the endosome contents. Third, we will isolate mutants that are resistant to the hybrid toxin. Fourth, we will biochemically and genetically characterize the mutants.